Purification, characterization, molecular cloning and extracellular production of a phospholipase A'1 from Streptomyces albidoflavus NA297

摘要

A novel metal ion-independent phospholipase A"1 of Streptomyces albidoflavus isolated from Japanese soil has been purified and characterized. The enzyme consists of a 33-residue N-terminal signal secretion sequence and a 269-residue mature protein with a deduced molecular weight of 27,199. Efficient and extracellular production of the recombinant enzyme was successfully achieved using Streptomyces lividans cells and an expression vector. A large amount (25 mg protein, 14.7 kU) of recombinant enzyme with high specific activity (588 U/mg protein) was purified by simple purification steps. The maximum activity was found at pH 7.2 and 50 ^oC. At pH 7.2, the enzyme preferably hydrolyzed phosphatidic acid and phosphatidylserine; however, the substrate specificity was dependent on the reaction pH. The enzyme hydrolyzed lysophosphatidylcholine and not triglyceride and the p-nitrophenyl ester of fatty acids. At the reaction equilibrium, the molar ratio of released free fatty acids (sn-1:sn-2) was 63:37. The hydrolysis of phosphatidic acid at 50 ^oC and pH 7.2 gave apparent V"m"a"x and k"c"a"t values of 1389 @mmol min^-^1 mg protein^-^1 and 630 s^-^1, respectively. The apparent K"m and k"c"a"t/K"m values were 2.38 mM and 265 mM^-^1 s^-^1, respectively. Mutagenesis analysis showed that Ser11 is essential for the catalytic function of the enzyme and the active site may include residues Ser216 and His218.